A variety of methods for forming an amino acid thiohydantoin (TH), either for use in C-terminal peptide sequencing, or for use in generating standards for C-terminal sequencing procedures, have been proposed. In one general method, the amino acid (or peptide) is activated at its carboxyl terminus with an anhydride, such as acetic anhydride, in the presence of a thiocyanate (ITC) salt or acid, to form a C-terminal peptidyl-TH via a C-terminal ITC intermediate (Stark, 1972). The peptidyl-TH can be cleaved to produce a shortened peptide and a C-terminal amino acid TH, which can be identified, e.g., by high pressure liquid chromatography (HPLC). The coupling conditions in this method typically require about 90 minutes at a 60.degree.-70.degree. C. (Meuth), and often lead to degradation of amino acid side chains in the peptide. For example, the side chain hydroxyl groups of serine and threonine may be attacked by the anhydride, requiring hydroxyl group protection.
A thiohydantoin formation method which can be carried out under milder conditions has been described by the inventor and co-workers (Hawke). Using trimethylsilyl isothiocyanate (TMS-ITC) as the reagent, TH formation was achieved by activation of the peptide with acetic anhydride for 15 min at 50.degree. C., followed by reaction with TMS-ITC for an additional 30 min at 50.degree. C. Despite the milder reaction conditions, the method nonetheless involves peptide exposure to a highly reactive anhydride activating agent, resulting in undesired side chain modifications.